Extracellular vesicles (EV),nanoscale vesicles encapsulated by phospholipid bilayers,are rich in biological molecules such as nucleic acids,metabolites,proteins,and lipids derived from parental cells.They are mainly involved in intercellular communication,signal transmission,and material transport and affect the functions of target cells.Ovulation disorders account for a higher proportion in the factors causing infertility which demonstrates increasing incidence year by year.Non-coding RNAs participate in a series of physiological and pathological processes of follicular development,playing a key role in female infertility.This review systematically introduces the types and biological roles of EV and elaborates on the regulation of follicular development from the effects of EV and non-coding RNAs on granulosa cell function,oocyte maturation,ovulation,luteal formation,and steroid hormone synthesis,providing a new idea and a breakthrough point for the diagnosis and treatment of infertility.
Female ; Humans ; Oogenesis/physiology* ; Granulosa Cells ; Extracellular Vesicles/physiology* ; Cell Communication ; RNA, Untranslated ; Infertility

Melatonin,an endocrine hormone synthesized by the pineal gland,plays an important role in the reproduction.The growth and development of follicles is the basis of female mammalian fertility.Follicles have a high concentration of melatonin.Melatonin receptors exist on ovarian granulosa cells,follicle cells,and oocytes.It regulates the growth and development of these cells and the maturation and atresia of follicles,affecting female fertility.This paper reviews the protective effects and regulatory mechanisms of melatonin on the development of ovarian follicles,granulosa cells,and oocytes and makes an outlook on the therapeutic potential of melatonin for ovarian injury,underpinning the clinical application of melatonin in the future.
Animals ; Female ; Melatonin/pharmacology* ; Ovarian Follicle ; Oocytes ; Granulosa Cells/physiology* ; Mammals

This study aims to observe the effect and explore the mechanism of Qirong Tablets in the treatment of premature ovarian insufficiency(POI) in mice via the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/hypoxia inducible factor 1(HIF-1) signaling pathway. Sixty SPF female BALB/c mice were randomly divided into normal group, model group, positive control group, Qirong Tablets low-, medium-and high-dose group. The normal group was intraperitoneally injected with the same amount of normal saline, and the other groups were intraperitoneally injected with cyclophosphamide 120 mg·kg~(-1)·d~(-1) once to establish a POI animal model. After the model was successfully established, the low-, medium-and high-dose groups of Qirong Tablets were administered orally with 0.6, 1.2, 2.4 mg·kg~(-1)·d~(-1) respectively. The positive control group was given 0.22 mg·kg~(-1)·d~(-1) Clementine Tablets by intragastric administration, and the normal group and model group were given intragastric administration with the same amount of normal saline, and the treatment was 28 d as a course of treatment. After drug intervention, enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of estradiol(E_2), follicle-stimulating hormone(FSH), luteinizing hormone(LH), and anti-mullerian hormone(AMH) in peripheral blood, and hematoxylin-eosin(HE) staining to observe the ovarian tissue. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay was used to detect the apoptosis of granulosa cells, and Western blot to determine the expression levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, PI3K, Akt, and HIF-1. Compared with the normal group, the modeling of POI caused loose or destroyed ovarian tissue with vacuolar structures, edema and fibrosis in the ovarian interstitium, disordered or loose arrangement of granulosa cells, and reduced normal follicles. Compared with the model group, drug interventions restored the ovarian tissue and follicles at all the development stages and reduced atretic follicles. Compared with the normal group, the modeling of POI lowered the serum level of E_2 and AMH(P<0.01), and elevated the level of FSH and LH(P<0.01). Compared with the model group, high-dose Qirong Tablets elevated the levels of E_2 and AMH(P<0.05), and lowered the levels of FSH and LH(P<0.05). Compared with the normal group, the modeling of POI up-regulated the protein levels of PI3K, Akt, HIF-1, Bax, and caspase-3 and down-regulated the protein level of Bcl-2 in the ovarian tissue(P<0.01). Compared with the model group, low-, medium-, and high-dose Qirong Tablets down-regulated the protein levels of PI3K, Akt, HIF-1, Bax, and caspase-3 proteins and up-regulated the protein level of Bcl-2 in the ovarian tissue(P<0.05). In conclusion, Qirong Tablets can up-regulate the expression Bcl-2, down-regulate the expression of Bax and caspase-3 in POI mice. Qirong Tablets may inhibit the apoptosis of follicular granulosa cells in mice, thereby delaying ovarian aging, improving reproductive axis function, and strengthening ovarian reserve capacity, which may be associated with the inhibition of PI3K/Akt/HIF-1 pathway.
Humans ; Mice ; Female ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; bcl-2-Associated X Protein ; Phosphatidylinositol 3-Kinases/metabolism* ; Caspase 3/metabolism* ; Saline Solution/therapeutic use* ; Signal Transduction ; Granulosa Cells ; Primary Ovarian Insufficiency/drug therapy* ; Follicle Stimulating Hormone/therapeutic use* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis

OBJECTIVES: Polycystic ovary syndrome (PCOS) is one of the most common endocrine diseases in women with reproductive age, which is associated with hyperandrogenism, insulin resistance, and ovulatory dysfunction. Progesterone receptor membrane component 1 (PGRMC1) can mediate progesterone to inhibit the apoptosis of ovarian granulosa cells and the growth of follicles, and to induce glucolipid metabolism disorder in ovarian granulosa cells, which is closely related to the occurrence and development of PCOS. This study aims to determine the expression of PGRMC1 in serum, ovarian tissue, ovarian granulosa cells, and follicular fluid in PCOS patients and non-PCOS patients, analyze the value of PGRMC1 in diagnosis and prognosis evaluation of PCOS, and investigate its molecular mechanism on ovarian granulosa cell apoptosis and glucolipid metabolism. METHODS: A total of 123 patients were collected from the Department of Obstetrics and Gynecology in Guangdong Women and Children Hospital (hereinafter referred to as "our hospital") from August 2021 to March 2022 and divided into 3 groups: a PCOS pre-treatment group (n=42), a PCOS treatment group (n=36), and a control group (n=45). The level of PGRMC1 in serum was detected by enzyme linked immunosorbent assay (ELISA). The diagnostic and prognostic value of PGRMC1 was evaluated in patients with PCOS by receiver operating characteristic (ROC) curve. Sixty patients who underwent a laparoscopic surgery from the Department of Obstetrics and Gynecology in our hospital from January 2014 to December 2016 were collected and divided into a PCOS group and a control group (n=30). The expression and distribution of PGRMC1 protein in ovarian tissues were detected by immunohistochemical staining. Twenty-two patients were collected from Reproductive Medicine Center in our hospital from December 2020 to March 2021, and they divided into a PCOS group and a control group (n=11). ELISA was used to detect the level of PGRMC1 in follicular fluid; real-time RT-PCR was used to detect the expression level of PGRMC1 mRNA in ovarian granulosa cells. Human ovarian granular cell line KGN cells were divided into a scrambled group which was transfected with small interfering RNA (siRNA) without interference and a siPGRMC1 group which was transfected with specific siRNA targeting PGRMC1. The apoptotic rate of KGN cells was detected by flow cytometry. The mRNA expression levels of PGRMC1, insulin receptor (INSR), glucose transporter 4 (GLUT4), very low density lipoprotein receptor (VLDLR), and low density lipoprotein receptor (LDLR) were determined by real-time RT-PCR. RESULTS: The serum level of PGRMC1 in the PCOS pre-treatment group was significantly higher than that in the control group (P<0.001), and the serum level of PGRMC1 in the PCOS treatment group was significantly lower than that in the PCOS pre-treatment group (P<0.001). The areas under curve (AUC) of PGRMC1 for the diagnosing and prognosis evaluation of PCOS were 0.923 and 0.893, respectively, and the cut-off values were 620.32 and 814.70 pg/mL, respectively. The positive staining was observed on both ovarian granulosa cells and ovarian stroma, which the staining was deepest in the ovarian granulosa cells. The average optical density of PGRMC1 in the PCOS group was significantly increased in ovarian tissue and ovarian granulosa cells than that in the control group (both P<0.05). Compared with the control group, the PGRMC1 expression levels in ovarian granulosa cells and follicular fluid in the PCOS group were significantly up-regulated (P<0.001 and P<0.01, respectively). Compared with the scrambled group, the apoptotic rate of ovarian granulosa cells was significantly increased in the siPGRMC1 group (P<0.01), the mRNA expression levels of PGRMC1 and INSR in the siPGRMC1 group were significantly down-regulated (P<0.001 and P<0.05, respectively), and the mRNA expression levels of GLUT4, VLDLR and LDLR were significantly up-regulated (all P<0.05). CONCLUSIONS Serum level of PGRMC1 is increased in PCOS patients, and decreased after standard treatment. PGRMC1 could be used as molecular marker for diagnosis and prognosis evaluation of PCOS. PGRMC1 mainly localizes in ovarian granulosa cells and might play a key role in regulating ovarian granulosa cell apoptosis and glycolipid metabolism.
Child ; Pregnancy ; Humans ; Female ; Polycystic Ovary Syndrome ; Apoptosis ; Granulosa Cells ; Lipid Metabolism ; Membrane Proteins ; Receptors, Progesterone

OBJECTIVE: To assess the association of gene expression with development potential of early embryos derived from patients with polycystic ovary syndrome (PCOS). METHODS: Three pairs of infertile patients with respectively matched age, body mass index, ovarian reserve and treatment with gonadotrophin-releasing hormone (GnRH) antagonists were selected. Patients with fewer embryos were assigned as the case group (n = 3), whilst the remainders were assigned as the control group (n = 3). Ovarian granulosa cells from patients were collected for the extraction of total RNA and subjected to RNA sequencing. The results were subjected to differential gene expression and functional enrichment analyses. RESULTS: Compared with the control group, 76 genes were up-regulated and 110 genes were down-regulated in the case group. The level of estradiol (E₂) was significantly higher in the control group on the trigger day with the injection of human chorionic gonadotrophin (HCG). Compared with the control group, the KRT7 gene was most significantly up-regulated, whilst the CCNYL2 gene was most significantly down-regulated in the case group. Gene ontology (GO) entries enrichment has found those associated with chromosome segregation, cell cycle regulation, and fatty acid metabolism to be significantly enriched. The genes participating in the regulation of cell assembly, differentiation, negative regulation of cell cycle, negative regulation of development, extracellular regulated protein kinases (ERK), ERK1 and ERK2 signaling pathways to be significantly down-regulated. KEGG enrichment analysis of cell signaling pathways revealed that steroid hormone biosynthesis-related genes were enriched. CONCLUSION Among patients treated with GnRH antagonists, the significant difference in the number of oocytes fertilized in vitro and the number of available embryos are associated with the difference in the expression of genes of ovarian granulosa cells.
Female ; Humans ; Pregnancy ; Embryonic Development ; Gene Expression ; Gonadotropin-Releasing Hormone/antagonists & inhibitors* ; Granulosa Cells ; Polycystic Ovary Syndrome/genetics*

OBJECTIVE: To investigate the role of tacrolimus-binding protein 38 (FKBP38) in follicle development and the mechanism by which Fkbp38 gene deletion causes premature ovarian insufficiency (POI). METHODS: The Cre-loxp system was used to construct oocyte-specific Fkbp38 knockout transgenic mice. The genotype of the transgenic mice was identified using PCR, and the expression of FKBP38 in the oocytes was verified. The numbers of primordial follicles, primary follicles, secondary follicles and antral follicles in Fkbp38 knockout mice and non-transgenic littermate control mice were counted with HE staining under a microscope for analyzing the effect of Fkbp38 deletion on follicular development. The fertility and serum sex hormone levels of the mice were determined by reproduction experiments and ELISA to assess ovarian function. Ovarian granulosa cell apoptosis of the mice was assessed using TUNEL assay. The activity of the downstream target protein of phosphorylated ribosomal S6 (PS6) of mTOR signaling pathway was detected, and the expressions of BCL-2 and BAX proteins were determined using immunofluorescence assay for assessing oocyte development in the mice. RESULTS: The oocyte-specific Fkbp38 knockout transgenic mouse model was successfully constructed, which showed decreased fertility, disordered sex hormone levels, and significantly reduced primordial follicles, primary follicles and secondary follicles in the ovary (P < 0.05), demonstrating POI-like changes. Compared with the control mice, oocyte-specific Fkbp38 knockout caused activation of the mTOR signaling pathway, significantly increased apoptosis of the granulosa cells, and obviously increased the BAX/BCL- 2 ratio by increasing BAX expression and reducing BCL-2 expression in the oocytes (P < 0.05). CONCLUSION FKBP38 plays an important role in follicle development, and Fkbp38 gene deletion in mice causes POI possibly by activating the mTOR signaling pathway and inducing granulosa cell apoptosis.
Female ; Humans ; Mice ; Animals ; Primary Ovarian Insufficiency/genetics* ; Apoptosis ; Signal Transduction ; Proto-Oncogene Proteins c-bcl-2 ; Granulosa Cells ; Mice, Transgenic ; Mice, Knockout ; TOR Serine-Threonine Kinases

OBJECTIVE: To investigate the effect of environmental estrogen bisphenol A (BPA) exposure on apoptosis of mouse ovarian preantral follicular granulosa cells and ovarian development and explore the underlying mechanism. METHODS: Mouse ovarian preantral follicular granulosa cells were isolated from female ICR mice at postnatal day (PND) 10 and cultured RESULTS: Compared with the control cells group, the isolated cells exposed to a low concentration of BPA (50 μmol/L) showed a significantly lowered apoptosis rate, increased mitochondrial membrane potential, and enhanced cellular proliferation ( CONCLUSIONS BPA can concentration-dependently regulate the function of ovarian preantral follicular granulosa cells in mice and potentially affects both the pregnant mice and the offspring female mice in light of early ovarian development.
Animals ; Apoptosis ; Benzhydryl Compounds ; Female ; Granulosa Cells ; Mice ; Mice, Inbred ICR ; Ovarian Follicle ; Phenols ; Pregnancy

In recent years,microRNAs(miRNAs)have been detected at different stages of follicular development and in different cells of follicles.Extracellular vesicle(EV)-derived miRNAs have also been detected in the follicular fluid of mature follicles.miRNAs participate in the regulation of normal follicular development,and the regulation disorder may lead to the occurrence of some ovarian diseases.In order to further systematically elucidate the regulatory mechanism of miRNAs on follicular development and find suitable EV-derived miRNAs that can predict oocyte development,we reviewed the functions of miRNAs in follicular development from the perspectives of granulosa cell development,oocyte development,and hormone synthesis.
Female ; Follicular Fluid ; Granulosa Cells ; Humans ; MicroRNAs/genetics* ; Oogenesis ; Ovarian Follicle

Extracellular vesicles (EVs) refer to bilayer membrane transport vesicles secreted by cells. EVs can take macromolecules from cells and transfer them to receptor cells. Among these macromolecular substances, the most studied are microRNAs (miRNAs). miRNA is non-coding RNA involved in the regulation of gene expression. It has been confirmed that there are different non-coding RNAs in mammalian follicular fluid EVs. EVs carrying miRNA can act as an alternative mechanism for autocrine and paracrine, affecting follicular development. This paper systematically introduced the kinds, characteristics and methods of isolation and identification of EVs, focusing on the effects of EVs and miRNAs on follicular development, including early follicular development, oocyte maturation, follicular dominance and effects on granulosa cell function. At the same time, the authors prospected the future research of EVs and microRNAs in follicular fluid, and provided ideas and directions for the research and application of EVs and miRNA functions in follicular fluid.
Animals ; Extracellular Vesicles ; metabolism ; Female ; Follicular Fluid ; chemistry ; Granulosa Cells ; drug effects ; MicroRNAs ; pharmacology ; Oogenesis ; drug effects

The aim of this paper was to observe the concentration,time and mechanism of autophagy induced by triptolide( TP) in ovarian granulosa cells( OGCs). CCK-8 method was used to compare the inhibitory effects of TP at different concentrations on primary cultured rat OGCs and IC50 was calculated. The effects of TP at different concentrations and time points on the expression of OGCs autophagy factor protein and the cascade of PI3 K/AKT/m TOR pathway were detected by Western blot. The effects of TP,autophagy inducer( brefeldin A) and PI3 K/m TOR inhibitor( NVP-BEZ235) on the expression of PI3 K/AKT/m TOR cascade and autophagy related factor protein were detected by Western blot. The results show that the IC50 of different concentrations of TP on OGCs of rat ovary was14. 65 μmol·L-1,and the minimum inhibitory concentration of TP was 0. 1 μmol·L-1( 100 nmol·L-1). Compared with the control group,the expression levels of beclin1 and LC3Ⅱ in each group were significantly higher than those in the control group( P<0. 05 or P<0. 01). After 12 hours of treatment with TP,brefeldin A and NVP-BEZ235,respectively,compared with the control group,TP could significantly promote the expression level of downstream autophagy effect or molecule beclin1,LC3Ⅱ and inhibit the expression level of LC3Ⅰ,p62 protein( P<0. 05 or P< 0. 01). Moreover,the expression of beclin1 and LC3Ⅱ/LC3Ⅰ in TP group was higher than that in brefeldin A group( P<0. 05 or P<0. 01),and the expression of p62 in TP group was lower than that in brefeldin A group( P<0. 05 or P<0. 01). At the same time,TP could significantly inhibit the expression of p-PI3 K,p-AKT,p-mTOR protein,and the inhibitory effect of TP was better than that of NVP-BEZ235 group. This study suggests that 100 nmol·L-1 TP could induce OGCs autophagy successfully in cultured rat ovary for 12 h; TP may induce OGCs autophagy by inhibiting PI3 k/Akt/m TOR signaling pathway.
Animals ; Apoptosis ; Autophagy ; Cell Proliferation ; Cells, Cultured ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Female ; Granulosa Cells ; drug effects ; Phenanthrenes ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Signal Transduction ; TOR Serine-Threonine Kinases ; metabolism